Plants have the largest mitochondrial (mt) genomes known, ranging in complexity from 300 to 2500 kb as compared to only 16kb in animal and 78kb in yeast mitochondria. In addition to this genetic complexity, plant mtDNAs are also physically complex. Unlike the mitochondrial genomes of animals and fungi which are generally found as a single size class, plant mtDNAs are found as a heterogeneous population of different size molecules present in varying concentrations. Whether this unique genome arrangement plays a role in plant mitochondrial gene regulation is unknown. However, it is possible that such a dispersed gene organization may be used by plants to amplify selectively specific mitochondrial genes in response to different stimuli. Among the many plant systems available tobacco has one of the simplest mitochondrial genomes and one of the best characterized plant cell suspension culture systems. Capitalizing on these features of the tobacco system we plan to construct a detailed physical map of the tobacco mitochondrial genome and to begin studying the mitochondrial genetics of tobacco in culture. For the tenure of this grant we plan to (1) identify and compare the origin(s) of replication of the different size classes; (2) isolate and map the RNAs coded for by each size class and translate the mRNAs in vitro; (3) using yeast mt genes, identify and map the homologous genes in tobacco; (4) isolate and characterize tobacco mitochondrial mutants; and (5) explore different possibilities for the creation of novel mitochondrial genomes. Out long term goals are to gain greater understanding of the organization, regulation and expression of the unique mitochondrial genome of higher plants. In addition these studies may also contribute to an understanding of the evolutionary pathways that have resulted in the different organizational strategies found in animal, fungal and plant mitochondrial genomes.